Transcriptional programming of cell identity is gaining importance at both the basic developmental and the clinical levels. While the phenomenology of cell programming and reprogramming by forced expression of transcription factors is well described, the mechanism(s) of action of programming factors or the sequence of regulatory events resulting in a cell adopting a new identity are largely unknown. We are combining the strengths of stem cell biology with genomic and computational approaches to map the process of transcriptional programming of spinal motor neuron (MN) identity at a deep molecular level. We have developed efficient methods for the induction of MN identity in differentiating embryonic stem cells (ESCs) by the expression of defined transcription factors. Using this system, we will combine biochemical, genomic, and computational analysis to address following questions: i) how is Isl1 recruited to transient enhancers in postmitotic motor neurons; ii) does Isl1 control enhancer activation; iii) are Klf/Sp factors bound to MN enhancers important for mediator and cohesin recruitment; iv) what motifs and factors coordinate interactions between distal and proximal MN- specific enhancers; v) can we infer the mechanisms controlling MN subtype specification and maturation by studying cell type and cell stage-specific regulatory regions in primary MNs. Together these studies will provide fundamental insight into the developmental processes underlying the specification of defined cell identity and will provide a novel and efficient source of MNs for disease modeling, functional analysis, and drug discovery.